Animal models for the study of hepatitis B virus infection

Even with an effective vaccine, an estimated 240 million people are chronically infected with hepatitis B virus(HBV) worldwide. Current antiviral therapies,including interferon and nucleot(s)ide analogues,rarely cure chronic hepatitis B. Animal models are very crucial for understanding the pathogenesis of chronic hepatitis B and developing new therapeutic drugs or strategies. HBV can only infect humans and chimpanzees, with the use of chimpanzees in HBV research strongly restricted. Thus, most advances in HBV research have been gained using mouse models with HBV replication or infection or models with HBV-related hepadnaviral infection. This review summarizes the animal models currently available for the study of HBV infection.     

Effect of concurrent acute infection with hepatitis C virus on acute hepatitis B virus infection.

OBJECTIVE--To investigate the possible interference with acute hepatitis B virus infection by co-infection with hepatitis C virus. DESIGN--Analysis of stored sera collected for transfusion transmitted viruses study in 1970s. SETTING--Four major medical centres in the United States. PATIENTS--12 recipients of blood infected with hepatitis B virus. MAIN OUTCOME MEASURES--In 1970s, presence of antibodies in hepatitis B virus and raised serum alanine aminotransferase concentration; detection of antibodies to hepatitis C virus with new enzyme linked immunoassays. RESULTS--Five of the 12 patients were coinfected with hepatitis C virus. Hepatitis B surface antigen was first detected at day 59 in patients infected with hepatitis B virus alone and at day 97 in those coinfected with hepatitis C virus (p = 0.01); median durations of antigenaemia were 83 and 21 days respectively (p = 0.05), and the antigen concentration was lower in the coinfected patients. Alanine aminotransferase patterns were uniphasic when hepatitis B virus infection occurred alone (range 479-2465 IU/l) and biphasic in patients with combined acute infection (no value > 380 IU/l; p = 0.0025). Four coinfected recipients developed chronic hepatitis C virus infection. The fifth patient was followed for only four months. CONCLUSIONS--Acute coinfection with hepatitis C virus and hepatitis B virus inhibits hepatitis B virus infection in humans, and onset of hepatitis B may reduce the severity of hepatitis C virus infection but not frequency of chronicity. Alanine aminotransferase concentration showed a biphasic pattern in dual infection.     

Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.     

Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees.

Of 13 different strains of hepatitis C virus (HCV) in the inoculum used, only 1 persisted in human lymphocyte cell lines infected in vitro (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). To determine whether that particular strain (designated H1-2) has a tropism for lymphocytes in vivo, we sequenced hypervariable region 1 (HVR1) of the genome of HCV recovered from the sera, livers, and peripheral blood mononuclear cells (PBMC) of chimpanzees infected with plasma H77, the same inoculum used for the in vitro studies. In the PBMC collected from two chimpanzees during the early phase of infection, H1-2 was detected as the only or predominant HVR1 sequence. H1-2 was also detected in PBMC obtained during persistent infection from a chimpanzee that had been treated with immunosuppressants. From the livers of these chimpanzees, two to six different strains were recovered but H1-2 was not detected. Thus, H1-2 appeared to have an affinity for lymphocytes not only in vitro but also in vivo. In samples collected from a chimpanzee after 6 years of infection, however, such tissue compartmentalization of the HCV genome was not observed; a single strain became predominant in the serum, liver, and PBMC. An HCV strain capable of replicating in both the liver and PBMC probably emerged during in vivo replication and persisted.     

Hepatitis delta antigen. Antigenic structure and humoral immune response

Hepatitis delta virus (HDV) is a small RNA virus that is dependent on helper functions provided by hepatitis B virus. The hepatitis delta Ag (HDAg) is the only protein known to be made from the viral genome, from an ORF with a coding capacity of 214 amino acids. The immunogenic epitopes of HDAg and the immune response to it were mapped by the use of synthetic peptides, antipeptide antibodies, and human mAb. Antipeptide sera covering approximately 60% of the linear sequence reacted with liver-derived HDAg. Antisera from HDV-infected humans, chimpanzees, and woodchucks reacted with from 2 to 13 of 15 peptides. The epitopes of two human anti-HD mAb were mapped to overlapping but distinct epitopes in the region around residues 106-123. Sera from infected humans, chimpanzees, and woodchucks were also tested by competition with the mAb. Use of the peptides and antipeptide sera defined one region in the sequence (residues 52-93) which is immunodominant in the immune response to HDAg. Reactivity of both peptides and antipeptide antibodies was very broad, covering most or all of the linear sequence. Competition assays also provided information on conformational epitopes, as well as the sequential epitopes defined by direct assays. The peptides and antipeptide antibodies should be useful in new assay development, in dissecting the anti-HD response in terms of chronic vs self-limited infection, and in studying the role of anti-HD in infection and recovery.     

Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.     

Simian Immunodeficiency Virus Infection of Chimpanzees (Pan troglodytes) Shares Features of Both Pathogenic and Non-pathogenic Lentiviral Infections

The virus-host relationship in simian immunodeficiency virus (SIV) infected chimpanzees is thought to be different from that found in other SIV infected African primates. However, studies of captive SIVcpz infected chimpanzees are limited. Previously, the natural SIVcpz infection of one chimpanzee, and the experimental infection of six chimpanzees was reported, with limited follow-up. Here, we present a long-term study of these seven animals, with a retrospective re-examination of the early stages of infection. The only clinical signs consistent with AIDS or AIDS associated disease was thrombocytopenia in two cases, associated with the development of anti-platelet antibodies. However, compared to uninfected and HIV-1 infected animals, SIVcpz infected animals had significantly lower levels of peripheral blood CD4+ T-cells. Despite this, levels of T-cell activation in chronic infection were not significantly elevated. In addition, while plasma levels of β2 microglobulin, neopterin and soluble TNF-related apoptosis inducing ligand (sTRAIL) were elevated in acute infection, these markers returned to near-normal levels in chronic infection, reminiscent of immune activation patterns in ‘natural host’ species. Furthermore, plasma soluble CD14 was not elevated in chronic infection. However, examination of the secondary lymphoid environment revealed persistent changes to the lymphoid structure, including follicular hyperplasia in SIVcpz infected animals. In addition, both SIV and HIV-1 infected chimpanzees showed increased levels of deposition of collagen and increased levels of Mx1 expression in the T-cell zones of the lymph node. The outcome of SIVcpz infection of captive chimpanzees therefore shares features of both non-pathogenic and pathogenic lentivirus infections.     

Immunogenicity of human hepatitis B virus P-gene derived proteins

The frequency and specificity of antibodies to P-gene encoded proteins of human hepatitis B virus was tested in sera of acute and chronically infected patients with and without hepatocellular carcinoma (HCC). For antibody detection an immunoprecipitation gel assay was performed with radioactively labeled polypeptides produced by in vitro translation of RNA of different P-gene regions. Thus, five antigenic regions were identified. All anti-P antibody positive sera reacted with carboxy-terminal P-poly-peptides, a subset with polypeptides of the amino-terminal and middle region, and none reacted with P-protein derived from the most sequence variable region. Anti-P antibodies were detected at very high frequency in sera of acute (73%) and chronically infected patients without HCC (87%), but less often in HCC patients (27%). These data indirectly demonstrate the expression of most hepatitis B virus P-gene sequences and the immunogenicity of P-proteins in vivo. Moreover, they establish hepatitis B virus anti-P-antibodies as a frequent serologic marker of infection and identify the carboxy-terminal region of the P-protein(s) as immunodominant.     

Tumor necrosis factor-alpha and interleukin-4 in the blood sera of chronic hepatitis patients

The concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) in the blood sera of chronic hepatitis patients was, on the average, reliably higher than in the control group. A more pronounced increase in the concentration of TNF-alpha in the blood sera was observed in patients with chronic hepatitis of viral etiology in comparison with toxic hepatitis. The pronounced cytokine response of type Th2, manifested by the excessive production of IL-4, was typical for hepatitis B virus and hepatitis C virus infections, but not characteristic of hepatitis D virus infection. The replication activity of hepatotropic viruses induced a powerful cytokine response. In the presence of active virus replication in patients with chronic hepatitis B the levels of both TNF-alpha and IL-4 in their blood sera proved to be reliably higher than in patients with hepatitis B virus in the interactive phase.     

Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection

The presence of the two "large" surface proteins of hepatitis B virus (HBV), P39 and GP42 of pre-S1-hepatitis B surface antigen, was assayed in the serum of an experimentally infected chimpanzee by using antibodies to a pre-S1-specific fusion protein synthesized in Escherichia coli. The immune response to pre-S1-hepatitis B surface antigen was monitored by using the pre-S1 fusion protein as an antigen. pre-S1 proteins were detected in the serum early in the course of infection and prevailed as long as hepatitis B surface antigen did, together with hepatitis B e antigen and viral DNA. Thus, the pre-S1 antigen can be considered a novel diagnostic marker for acute HBV infection. Antibodies to pre-S1, both immunoglobulin M and G classes, were also detected early in infection, shortly after the appearance of the pre-S1 antigen, suggesting its strong immunogenicity in vivo. The anti-pre-S1 antibodies therefore also represent an early serological marker for acute HBV infection and, owing to their early appearance and persistence, may play a role in the neutralization of the virus.     

Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.     

Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan

Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.     

Antibody to hepatitis B core antigen in chronic active hepatitis.

Antibody to hepatitis B core antigen (anti-HBc), which has been assumed to be a more sensitive indicator of hepatitis B virus replication than hepatitis B surface antigen (HBsAg), was detected in the sera of 26 of our 65 patients with HBsAg-negative chronic active hepatitis. Thus despite the absence of HBsAg the liver disease could be the consequence of chronic infection with hepatitis B virus in these patients. They differed, however, from a group of 35 patients with HBsAg-positive hepatitis in being older on average and having less active liver lesions. The two groups could represent either two stages of chronic infection with hepatitis B virus or two types of response to it.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.     

Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.     

Additive effect of neutralizing antibody and antiviral drug treatment in preventing virus escape and persistence

Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus.     

Hepatitis B Virus Infection—Current Concepts of Chronicity and Immunity

Among the three types of viral hepatitis agents—A, B and non-A, non-B—the hepatitis B virus (HBV) has been best characterized by immunologic and recombinant DNA technologies. The indefinite persistence of hepatitis B virus infection in 85% to 90% of perinatally infected infants and in about 10% of those infected later in life accounts for a worldwide epidemiologic reservoir of more than 200 million carriers who are at a high risk for the development of δ-infection, chronic liver disease and hepatocellular carcinoma. Active immunization with a safe and effective vaccine, derived from the plasma of carriers of hepatitis B surface antigen (HBsAg), is envisaged to avoid viral hepatitis type B and its chronic sequelae. In addition to serologic and immunohistochemical markers of hepatitis B virus infection, hybridization assays using cloned HBV DNA have provided new insight into the biology of this virus, its persistence and its oncogenic potential in humans and in animal models. Genetic similarities have been recognized between HBV and the antigenically distinct non-A, non-B agents implicated in some cases of transfusion-associated chronic hepatitis. Structurally this unique group of HBV and HBV-like agents are DNA viruses with functional attributes of integration and replication analogous to the retroviruses.     

病毒性肝炎患者抗—HBcIgA 、IgE 、IgG 、IgM 平行检测的临床意义

目的：通过对乙肝病人血清抗-HBcIgA、HBcIgE、HbcIgc、HbcIgM平行检测进一步地探讨其临床应用价值。方法：收集216份乙肝病人血清（均为我院住院病人的血清）,采用ELISA法对病人血清抗-HBcIgA、HBcIgE、HBcIgc、HBcIgM平行检测,并对检测结果作统计学分析。结果：检测结果表明：抗-HBcIgA是HBV感染后肝脏损害的明显标志,抗-HBdgE是HBV感染后乙型肝炎慢性化的标志,抗-HBcIgG持续存在则是以往感染的标志,抗-HBcIgM是HBV感染后病毒持续复制的标志。结论：在急慢性乙型肝炎、慢活肝与慢迁肝鉴别诊断中,平行检测病人血清抗-HBcIgA、HBcIgE、HbcIgG、HbcIgM对乙型肝炎慢性化及其预后估价均有重要临床意义。     

IL-10, T cell exhaustion and viral persistence

Viral infections can have one of two outcomes: control of viral replication and acute infection or viral persistence and chronic infection. It is clear that both pathogen and host characteristics influence the acute versus chronic outcome of viral infection. The early events in the host immune response that favor immunosuppression and viral persistence, however, have remained poorly understood. Using the well-characterized mouse model of acute versus chronic lymphocytic choriomeningitis virus (LCMV) infection, two groups have recently identified the interleukin-10 (IL-10)/IL-10R pathway as a key regulator of acute versus chronic infection. Blockade of IL-10R converted a chronic LCMV infection into a rapidly controlled acute viral infection and prevented the functional exhaustion of memory T cells. These insights into the role of IL-10 in the establishment of chronic infection could lead to new therapeutic opportunities during human infections with pathogens such as HIV, hepatitis C virus (HCV) and hepatitis B virus (HBV).     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.